Fermentation agent



Patented Apr. 5, 1938 PATENT QOFFICE' FERMENTATION AGENT Rafael Arroyo,Rio Piedras, P. R., assignor, by mesne assignments, to The LnmmusCompany, v New York, N. Y., a corporation of Delaware No Drawing.Original application March 13,

1934, SeriaLNo. 715,374.

Divided and this application April 20, 1936, Serial No. 75,49!

'8 Glaims.

The present invention relates to the art of obtaining butanol andacetone by a fermentation procedure, in which the fermentable substratemay be produced directly from a final sugarhouse molasses and in which ahigh yield of butanol is obtained. This invention relates to thefermentation agent employed for this purpose, and is a division of mycopending application Serial No. 715,374, filed March 13, 1934.

'While it is known to obtain butanol and acetone by the fermentation ofstarches; as developed by Fernbach, Weizmann and others, and asdesecribed in the Weizmann Patents 1,315,585; 1,329,214; and 1,437,697;it has been found that the bacteria are highly selective in theiraction, and the procedures require a mash of cereal or nutty nature; andthat the bacteria do not opcrate upon the constituents of molasses. The

Robinson "Patent 1,510,526 describes a procedure in which the bacteriaare developed and cultivated upon a substrate of corn or other cerealmash, and molasses is added during the course of this normalfermentation, the patentee pointing out the poisoning or toxic effect ofthe molasses with respect to the bacteria employed: 1. e.

molasses alone is not a proper inoculum. for such bacteria.

It has now been found, however, that a simple and direct fermentation ofmolasses may be accomplished, with a yield much higher than that of thebacteria described in the prior art, by employing a particular type ofbacteria for this purpose.

Name: B. tetryl (new species).

I. Morphology 1. Vegetative cells motile in early stages, mo-

tility becoming less pronounced as the culture becomes older, andfinally is lost almost entirely in old cultures. Facultativelyanaerobic.

Medic used: Molasses of 4-6" Brix density;

the sucrose of which is inverted by the addition of sulphuric acid atthe rate of 1.5 c. c. conc. acid to 1800 c. c. of mash. It is thenneutralized, with CaCO: at rate of 5.5 grams 4 per above volume, andNH4OH added at rate 50. of 1.5 c. 0. Temp. of incubation 35 0, age 24Source: l rom roots of the Kassoer variety of hrs. Colonies thenestablished by plating on malt gelatine agar on inverted plates.Acclimatable to densities in excess of 12 Brix.

Form: Rods varying in length, occurring singly and in short and longchains. Peritrichous flagella.

Size: 1.5 x 5.6 microns. Ends'rounded.

Stain: Stain readily with gentian violet or methylene blue or carbonfuchsine. Gram positive. 10

2. Sporangia: Media used-6 Brier inverted molasses sol, asabovedescribed.

Form: Elliptical. 5

Spores: Polar, and resistant to ordinary methods used for stainingvegetative cells. Forms endospores in almost all media: while organismstill motile.

II. Cultural features 1. Slightly raised growth on malt gelatine agarslants after 24 hrs. at 35 0.; facultative anaerobe; nonliquefylnggelatine. v

In malt gelatine agar stabs growth filiform. 25 Surface growth on agar;scanty on agar stroke; dull in luster; smooth surface. Agar coloniescircular, diameter undetermined.

2. Good growth on potato plugs at 35 C., produces gas and foam whichsometimes rise above the plug.

Good growth in malt agar with calcium carbonate; and in potato dextrosesugar stabs; at first filiform growth, later splitting the agar andbecomes spreading with gas formation. Gas 35 from glucose and acidculture; gas from sucrose culture under methods and on medium prescribedby American Bacteriological Society, but apparently only because ofpresence of sugar inverted by heating during sterilization.

Little growth: 111 media without sugars. No growth: Cohns solution.Dunhams solution, NO: peptone solution.

but rennet curding absent: peptonization presm cut and positive: indoleproduction negative.

III. Physical and biochemical features 1. Fermentation tubes: Substancesfermented with gas evolution.

gal-absent, x-present, xii-strong, xxx-very strong, xxx-strongesFurther, cultures in mannose, salicin, galactose,

inositol, melezitose, glycogen and sorbitol gave negative results at theend of six days; while cultures in l-arabinose, inulin, and fructosegave positive acid (xxx) and gas (xxx) indications in three to fourdays.

Butanol (butyl alcohol), acetone and a small quantity of ethanol (but noiso-propanol) are produced in the fermentation of the lower sugars(monoses) but the organism is incapable of producing these products fromsucrose, or to hydrolyze sucrose into invert sugars. The gases formedare carbon dioxide and hydrogen. B. tetryl grows in starchy mashes, butdoes not produce either butanol or acetone to any appreciable degreetherein.

Bacillus tetryl is found in nature in the rind of the lower nodes ofsugar cane of the Kassoer variety grown in Puerto Rico. Direct culturesin sterile mash of 5 Brix were incubated, and colonies established byplating on malt gelatine agar on inverted plates.

Propagation from the colonies, by the use of serile molasses mash ofsuccessively increasing densities, has shown that the B. tetryl isacclimatable to densities in excess of 12 Brix.

As pointed out in the aforesaid copending application, a preferredprocedure in fermenting molasses, to obtain butanol and acetone,comprises the solution of "black strap final sugarhouse cane molasses(for example, about-88 Brix; polarization27.85; apparent purity-31.64;total sugars as invert5 1.50) in water, by heating on a water bath withagitation, and in the proportion of 220 grams of molasses to 400 cc. ofwater. 3.0 cc. of strong sulphuric acid is added and the mixtureautoclaved for thirty minutes at 20 pounds pressure to effect a completeinversion of the sucrose contained in the molasses. After cooling toabout 50 degrees C., 12 grams of calcium carbonate and 3.0 cc. of strong(20 percent) ammonia water are added to the mash. When effervescencesubsides, the mash is com pleted with distilled water according to thesugar concentration desired (for example, 1800 cc.) and sterilized byautoclaving for 30 minutes at 20 pounds pressure. When thus prepared,this standard mash gives a pH value of 6. It has been found desirable toadjust to the pH value of 6 by the addition of N/l00 sulphuric acid orN/ sodium hydroxide as necessary.

To this mash is preferably added from one to five grams per 1000 cc. ofan activating agent such as kieselguhr, bone black, lamp black oractivated carbon. Such an activating agent, as described and claimedherein, is itself substantial- 1y inert chemically, but is a solid in as ate of fine subdivision and has a large surface and a recognizedadsorption power; and its action appears to be physical rather thanchemical and to be of catalytic type. When lamp black is used alone, amaximum effect is'obtained by using two grams. When kieselguhr is usedalone, a maximum effect is obtained by using one or two grams.

The apparent absolute maximum is obtained by using two grams ofkieselguhr with two grams of lamp black. While it is possible to operatewithout the employment of an activating agent, the use of lamp blackalone led to an increase of .over fifteen per cent in yield; the use ofkieselguhr alone of over 12 percent; while the use of the two activatorstogether gives an increase of nearly 22 percent over the resultsattained when no activating agents were employed. Such activatorsoperateto enable an increase in the density and sugars concentration of themash, and to shorten the time of the fermentation period. An increase of20 percent in yield, 24 percent in concentration, and a reduction of 30percent in time can be obtained with activated carbon, for example.

The prepared mash is then seeded with Bacillus tetryl which has beenacclimatized to the density of the mash so that preferably it isisotonic therewith, and propagation effected at between 30 and 39degrees C. At the lower temperature, a greater length of time ofpropagation is required. At the higher temperature, an intense activityoccurs for a short time and then further action ceases prior toexhaustion of the substrate. The preferred range of temperature isbetween 32 and 37 degrees and the optimum yield of total solvents isattained at 35 degrees C. At temperatures from 35 to 37 degrees 0., thequantity of acetone increases while the quantity of butanol decreases.

At the preferred temperature of 35 degrees 0., about 24 to 48 hours arerequired for the fermentation. j

It has been found that the acidity of the mash has a striking part indetermining the condition of the operation. The Bacillus tetryl worksbest under slightly acid conditions, and the optimum pH of the mash isinitially around 6 (corresponding to a, potentiometer reading of 90millivolts with-a quinhydrone electrode). During normal fermentation,creases steadily during the first 22 or 24 hours to a reading of about160 millivolts (pH value of 5.0) Thereafter, the value decreases to anearly constant value of around to millivolts (pH value of 5.5), whichremained steady until fermentation stopped. In particular, it is foundthat in the event of contamination, or the presence of any inhibitingforce, the acidity continues to rise, and values as highas to 210millivolts have been found. The periodic determination of acidity,therefore, furnishes an excellent control for supervising the course ofthe action and determining the presence of impurities or undesiredfactors.

It has been found that the optimum concentration of total sugars formaximum yields is between 4.50 grams and 6.00 grams per hundred cc. Withincreasing concentrations, a greater time is required for fermentationat 35 degrees C., ranging from 48 to 52 hours: and yielding 24.75 partsof total solvents (butanol, acetone and ethanol) per 100 parts of sugarsat a sugar concentration of 3.65; 27.74 at a sugar concentration of4.25; 27.30 at a sugar concentration of 4.55; 27.12 at a sugarconcentration of 4.85; and 2.85 at a sugar concentration of 5.14. Atconcentrations of 50 the potentiometer reading in-- stances in the ratioof 14:4:1.

grams per hundred cc. and over, no fermentation appeared to occur. Theconcentration of 4.85 percent sugar is to be preferred when noactivators are used; but when activators are used, then concentrationsof from 5.5 to 6.0 percent sugars are preferable. On a commercial basisit is therefore preferred to operate with a concentration of about 4.85grams of total sugar per hundred cc. of mash, which has been found toyield on distillation 1,315 grams of total solvents or about 1.65 cc.Slightly lower sugar concentrations give higher yields per unit ofsugar, but the increased plant capacity for fermentation anddistillation involves cost factor differentials which render such lowerconcentrations uneconomical.

The total length of time for the fermentation, of course, depends uponthe several factors as above indicated. At a temperature of 30 degrees(3., hours were required to obtain 23.50 percent of total solvents; at32 degrees, 63 hours were required to obtain 24.50 percent; at 35degrees, 49 hours were required to obtain 29.50 percent; and at 37degrees, 38 hours were required to obtain 24.49 percent. As indicatedabove, 35 degrees C.

is regarded as the optimum temperature of prop-.

agation and fermentation.

When the fermentation is completed, the butanol, acetone and ethanol maybe distilled and recovered in the usual way.

The above examples are set out as a manner of proceeding with thefermentation agent of the present invention, and the method involved isdisclosed and claimed in the aforesaid copending application. Theparticular dilutions, acid additions, and alkali additions to adjust thepH value will depend upon the strength of the initial material in totalsugars, in sucrose, and in the general Brix density thereof; but aperson skilled in the fermentation art can determine the dilutions andadditions to be used and made, in accordance with the aboverecommendations.

A characteristic of the action of Bacillus tetryl upon invert sugars isthe high ratio of butanol obtained thereby. While the bacillus used inthe Weizmann process produces butanol, acetone and ethanol in the ratioof 6:3:1, and does not produce butanol or acetone from molasses at all,it is found that Bacillus tetryl produces these sub- For example, the

Total solvents 30.00% or 1.95 pounds Butanol 22.31% or 1.45 poundsAcetone 6.15% or 0.40 pounds Ethanol 1.54% or 0.10 pounds The capacityof Bacillus tetryl to produce a large quantity of butanol and anextremely low quantity of ethanol is striking.

It will be understood that the above statements with respect to theemployment of the above type of organism in effecting the conversion ofthe sugars of molasses to butanol and acetone are not restrictive, butthat the fermentation agent may be employed in other ways withoutdeparting from the scope of the appended claims.

I claim:

1. A culture of Bacillus tetryl in a substrate having a concentration ofinvert sugar between 3 to 6 grams per hundred cubic centimeters.

2. A culture of Bacillus tetryl in a substrate having a concentration ofinvert sugar between 4.50 and 6.00 grams per hundred cubic centimeters.

3. A culture of Bacillus tetryl in and acclimated to a substrate havinga concentration of invert.

sugar between 4.50 and 6.00 grams per hundred cubic centimeters, andbeing effective to produce a yield of substantially percent of theweight of fermented invert sugar as a mixture of butanol, acetone andethanol in the proportion of substantially 14:421.

4. A culture of Bacillus tetryl in a substrate having a concentration ofinvert sugar of substantially 4.85 grams per hundred cubic centimeters.

5. A culture of Bacillus tetryl in a dilute molasses substratecontaining invert sugar.

6. A culture of Bacillus tetryl in a dilute flnal' sugarhouse molassessubstrate having a pH of about 6 and containing invert sugar.

7. A culture for accomplishing fermentation of monose sugars forproducing butanol and acetone, consisting of Bacillus tetryl in asubstrate including an invert sugar and characterized in effecting theproduction from dextrose and levulose of butanol, acetone and ethanol inthe proportions of substantially 14:4:1.

RAFAEL ARROYO.

